title: The homoeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle.
abstract: OBJECTIVE
  Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fibre-type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown.
  
  
  METHODS
  The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14 days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2C12 mouse myoblasts were grown in standard conditions. Cells were cotransfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4 days of differentiation.
  
  
  RESULTS
  In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8-fold (±0.1, P < 0.05). With decreased Six1, MHC IIB expression decreased 2.7-fold (±0.7, P = 0.04). Proportion of muscle fibres expressing MHC IIB decreased (45.3 ± 4.8% vs. 65.1 ± 7.3% in control group, P = 0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6 ± 4.3% vs. 75.2 ± 5.4% in control group, P < 0.05). Decreased Six1 increased MHC IIA expression 1.9-fold (±0.3, P = 0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9-fold (±0.3, P < 0.01). Six1 knockdown repressed MHC IIB promoter 2.9-fold (±0.1, P < 0.05) and MHC IIX 3.7-fold (±0.08, P < 0.01).
  
  
  CONCLUSIONS
  Six1 knockdown caused a fast-to-slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fibre phenotype.
authors.affiliation: Department of Exercise Science, University of South Carolina, Columbia, SC, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Hetzler
authors.forename: B C
authors.initials: BC
authors.lastname: Collins
authors.forename: R A
authors.initials: RA
authors.lastname: Shanely
authors.forename: H
authors.initials: H
authors.lastname: Sue
authors.forename: M C
authors.initials: MC
authors.lastname: Kostek
mesh_terms: D000818:Animals; D015153:Blotting, Western; D018274:Electroporation; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D018398:Homeodomain Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis*; D020033:Protein Isoforms / Q000096:biosynthesis; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D018398:Homeodomain Proteins; D020033:Protein Isoforms; C490947:Six1 protein, mouse; D018995:Myosin Heavy Chains
keywords: Six1; myosin heavy chain; skeletal muscle